TY - JOUR
T1 - Structural determination of biomolecular interfaces by nuclear magnetic resonance of proteins with reduced proton density
AU - Ferrage, Fabien
AU - Dutta, Kaushik
AU - Shekhtman, Alexander
AU - Cowburn, David
N1 - Funding Information:
Acknowledgments We thank Geoffrey Bodenhausen for his careful reading of a version of the manuscript. Supported by grant GM 47021 from the National Institute of Health. AS was supported by grant 1-06-CD-23 from the American Diabetes Association. FF thanks the French Ministry of Foreign Affairs for a Lavoisier fellowship.
PY - 2010/5
Y1 - 2010/5
N2 - Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a protein) with high proton density to a target protein (or other biomolecule) with low proton density by using isotope-filtered nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein or protein-ligand docking. Simulation indicates significant gains in sensitivity because of the resultant relaxation properties, and the utility of this technique, including a method for direct determination of the protein interface, is demonstrated for two different protein-protein complexes.
AB - Protein interactions are important for understanding many molecular mechanisms underlying cellular processes. So far, interfaces between interacting proteins have been characterized by NMR spectroscopy mostly by using chemical shift perturbations and cross-saturation via intermolecular cross-relaxation. Although powerful, these techniques cannot provide unambiguous estimates of intermolecular distances between interacting proteins. Here, we present an alternative approach, called REDSPRINT (REDduced/Standard PRoton density INTerface identification), to map protein interfaces with greater accuracy by using multiple NMR probes. Our approach is based on monitoring the cross-relaxation from a source protein (or from an arbitrary ligand that need not be a protein) with high proton density to a target protein (or other biomolecule) with low proton density by using isotope-filtered nuclear Overhauser spectroscopy (NOESY). This methodology uses different isotropic labeling for the source and target proteins to identify the source-target interface and also determine the proton density of the source protein at the interface for protein-protein or protein-ligand docking. Simulation indicates significant gains in sensitivity because of the resultant relaxation properties, and the utility of this technique, including a method for direct determination of the protein interface, is demonstrated for two different protein-protein complexes.
KW - Cross-relaxation
KW - Csk SH3
KW - Isotope-filtered NOESY
KW - Partial deuteration
KW - Protein complex
KW - Ubiquitin
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U2 - 10.1007/s10858-010-9409-9
DO - 10.1007/s10858-010-9409-9
M3 - Article
C2 - 20372977
AN - SCOPUS:77951665540
SN - 0925-2738
VL - 47
SP - 41
EP - 54
JO - Journal of Biomolecular NMR
JF - Journal of Biomolecular NMR
IS - 1
ER -